| Maize (Zea mays L.) is one of the most widely planted crops worldwide. Maize is susceptible to chilling injury when sowed in early spring. Chilling severely inhibited seed germination and seedling growth, and eventually reduced yield. Therefore, it is important to elevate chilling tolerance of maize. As an important plant hormone, salicylic acid (SA) played a key role in regulating plant chilling tolerance. In present study, two maize inbred lines, Huang C (chilling-tolerant) and Mo17(chilling-sensitive) were used to investigate the regulation role of SA in seeds and seedlings of maize and its relationship with chilling tolerance. The main results were as follows:The changes of endogenous SA, antioxidant enzyme activities and malondialdehyde (MDA) concentrations were investigated in two maize inbred lines under chilling stress. The results showed that both endogenous free and bound salicylic acid contents were increased in roots and leaves of both lines. MDA concentrations were also increased significantly in roots and leaves of both lines after chilling stress. Besides, in Huang C, chilling stress increased the activities of four antioxidant enzymes, ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and peroxidase (POD); while in Mo17, only CAT and APX were increased. Furthermore, a stepwise regression analysis was conducted between SA and MDA concentrations or antioxidant enzyme activities under chilling stress. The results indicated that MDA concentrations were positively correlated with total SA contents in roots (R2=0.9557,P=0.0224) and bound SA in leaves (R2=0.9948, P=0.0458), respectively. Total SA contents had positive correlations with APX activities both in roots (R2=0.9986,P=0.002) and leaves (R2=0.9274, P=0.037) and GR in leaves (R2=0.8645, P=0.0221). Together, these results suggested that chilling stress improved the biosynthesis of endogenous SA, and lipid peroxidation and antioxidant enzymes activities could be indicated by endogenous SA contents of maize seedlings under chilling stress. Besides, increased activities of antioxidant enzymes, especially in roots, may contribute to the chilling tolerance of maize seedlings.One kind of biologically active SA analogue (acetylsalicylic acid, ASA) and two inactive compounds (4-aminosalicylic acid and4-aminobenzoic acid), along with SA were chosen to evaluate their role in inducing chilling tolerance of maize. These compounds were applied as seed treatments or as a hydroponic application. The results showed that four compounds had no significant effect on germination of maize seeds; however. SA or ASA soaking treatments significantly increased the root length, shoot height, shoot and root dry weights of seedlings grown under chilling stress. Hydroponic applications of SA or ASA significantly alleviated the accumulation of malondialdehyde, hydrogen peroxide and superoxide radicals in roots and leaves of both lines under chilling stress, and increased the photo synthetic pigments, including chlorophyll a, chlorophyll b and carotenoids. However,4-aminosalicylic acid and4-aminobenzoic acid applications had no significant effect in ameliorating the growth inhibition of seedlings under chilling stress. This study showed that SA and ASA significantly induced the chilling tolerance of maize; however,4-aminosalicylic acid and4-aminobenzoic acid were not effective in inducing tolerance to chilling stress. The results suggest that only SA analogues with biological activity may have the ability to induce chilling tolerance of maize.The effects of exogenously applied SA on physiological characteristics and catalase genes expression were investigated in two maize inbred lines under chilling stress. The results showed that hydroponic treatment with0.5mM SA significantly alleviated the accumulation of malondialdehyde and hydrogen peroxide in leaves and roots of both lines under chilling stress. Besides, the activities of CAT, APX and SOD were significantly increased by SA pretreatment in maize seedlings under chilling stress, while SA application decreased POD activity in those seedlings. Three Cat genes responded differentially to SA application in leaves and roots of seedlings after chilling stress. Cat] gene expression was improved by SA treatment both in leaves and roots of Mo17and conversely, the gene expression was inhibited in those of Huang C. Notably, SA application significantly enhanced Cat2gene expression in both leaves and roots of two maize lines. However, Cat3gene expression was inconsistent between leaves and roots of two lines responding SA treatment. The fresh and dry weights of seedlings were significantly increased by SA treatment under chilling stress. Overall, our results indicated that exogenous SA application increased tolerance of maize plants to chilling stress, mainly by enhancing the activities of APX, SOD and CAT and related Cat2gene expression.The effect of exogenously applied SA and/or SA biosynthesis inhibitors on endogenous SA contents, lipid peroxidation, antioxidant enzyme activities and growth of maize seedlings were investigated under chilling stress. The results showed that SA pretreatment significantly decreased MDA concentrations, improved antioxidant enzyme (i.e. SOD, CAT, POD and APX) activities, phenylalanine ammonia-lyase (PAL) and benzoic acid2-hydroxylase (BA2H) activities, increased PAL gene transcription of seedlings under chilling stress and endogenous SA contents in maize seedlings. Besides, the growth inhibition of seedlings under chilling stress was also alleviated by SA treatment. However, SA biosynthesis inhibitors, i.e.2-amino-2-indanophosphonic acid (AIP) and1-aminobenzotriazole (ABT) applications significantly increased MDA concentrations and SA contents, decreased antioxidant enzyme activities, PAL and BA2H activities and PAL gene transcription in leaves of maize seedlings under chilling stress. These inhibitor treatments further augmented the retardation of seedlings growth under stress. In comparison with inhibitor treatments, SA and inhibitor applications decreased MDA concenrtrations, improved enzyme activities and seedling growth status; which indicated that exogenous application of SA could recover the damage effect on seedlings by inhibitor treatments. Present study indicated that exogenous SA application induced chilling tolerance of maize seedlings, and the SA synthesized by PAL and BA2H participates in the acquisition of chilling tolerance of maize seedlings.The effects of exogenous application of SA, putrescine (Put). SA biosynthesis inhibitors and polyamines (PAs) biosynthesis inhibitors on endogenous SA and PAs contents, and the growth of seedlings were investigated in two maize inbred lines under chilling stress. The results showed that Put treatment significantly increased endogenous SA contents in leaves of seedlings, while PAs biosynthesis inhibitors, i.e. difluoromethylormithine (DFMO) and D-arginine (D-Arg) treatments significantly decreased SA contents. Besides, SA and Put application significantly improved Put contents in leaves of seedlings; AIP treatment increased endogenous Put contents of both maize lines, while ABT treatment only increased Put contents of Mo17. DFMO and D-Arg treatments significantly decreased Put and spermidine (Spd) contents of both lines. Application of SA and Put significantly decreased endogenous Spd contents of both lines under chilling stress; however, they increased Spd contents in seedlings after recovery growth under normal temperature. Moreover, application of SA and Put significantly alleviated the retardation of seedlings growth under chilling stress, while four inhibitors treatments aggravated the effect of chilling stress on seedlings growth. In conclusion, it suggested that Put application increased endogenous SA accumulation of maize seedlings under chilling stress, PAs inhibitors inhibited Put accumulation, thus decreased SA contents of seedlings. Besides, exogenous application of SA and Put could induce Put accumulation of seedlings, then Put was converted to Spd; therefore Put and Spd contents were increased finally. Present study indicated that chilling tolerance of maize seedlings can be induced by exogenous application of SA and Put, and there was a dual directional regulation mechanism between SA and Put in maize seedlings. |
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